Ionizable cationic lipid for rna delivery

ABSTRACT

What is described is a compound 
     
       
         
         
             
             
         
       
     
     wherein
         R 1  and R 2  are the same or different, each a linear or branched alkyl with 1-9 carbons, or an alkenyl or alkynyl with 2 to 11 carbons;   L 1  and L 2  are the same or different, each a linear alkyl having 3 to 18 carbons;   X 1  and X 3  are —CO—O—;   X 3  is S or O;   L 3  is a lower alkyl;   R 3  is a lower alkyl; and   R 4  and R 5  are the same or different, each a lower alkyl;
 
or a pharmaceutically acceptable salt thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/546,105, filed Nov. 18, 2014, which claims benefit under 35 U.S.C.§119(e) of Provisional U.S. patent application No. 61/905,724, filedNov. 18, 2013, the contents of which are incorporated herein byreference in its entirety.

BACKGROUND

A number of different types of nucleic acids are currently beingdeveloped as therapeutics for the treatment of a number of diseases.These nucleic acids include DNA in gene therapy, plasmids-basedinterfering nucleic acids, small interfering nucleic acids for use inRNA interference (RNAi), including siRNA, miRNA, antisense molecules,ribozymes and aptamers. As these molecules are being developed, therehas been developed a need to produce them in a form that is stable andhas a long shelf-life and that can be easily incorporated into ananhydrous organic or anhydrous polar aprotic solvent to enableencapsulations of the nucleic acids without the side-reactions that canoccur in a polar aqueous solution or nonpolar solvents.

The present invention relates to novel lipid compositions thatfacilitate the intracellular delivery of biologically active andtherapeutic molecules. The present invention relates also topharmaceutical compositions that comprise such lipid compositions, andthat are useful to deliver therapeutically effective amounts ofbiologically active molecules into the cells of patients.

The delivery of a therapeutic compound to a subject is important for itstherapeutic effects and usually it can be impeded by limited ability ofthe compound to reach targeted cells and tissues. Improvement of suchcompounds to enter the targeted cells of tissues by a variety of themeans of delivery is crucial. The present invention relates the novellipids, incompositions and methods for preparation that facilitate thetargeted intracellular delivery of biological active molecules.

Examples of biologically active molecules for which effective targetingto a patient's tissues is often not achieved include: (1) numerousproteins including immunoglobin proteins, (2) polynucleotides such asgenomic DNA, cDNA, or mRNA (3) antisense polynucleotides; and (4) manylow molecular weight compounds, whether synthetic or naturallyoccurring, such as the peptide hormones and antibiotics.

One of the fundamental challenges now facing medical practitioners isthat a number of different types of nucleic acids are currently beingdeveloped as therapeutics for the treatment of a number of diseases.These nucleic acids include DNA in gene therapy, plasmids smallinterfering nucleic acids (iNA) for use in RNA interference (RNAi),antisense molecules, ribozymes, antagomirs, microRNA and aptamers. Asthese nucleic are being developed, there is a need to produce lipidformulations that are easy to make and can be readily delivered to atarget tissue.

SUMMARY

What is described herein is a compound of Formula 1

in which

-   -   R₁ and R₂ are the same or different, each a linear or branched        alkyl of 1 to 9 carbons, an alkenyl or alkynyl of 2 to 11        carbons, or cholesteryl,    -   L₁ and L₂ are the same or different, each a linear alkylene or        alkenylene of 3 or 4 carbons,    -   X₁ is —CO—O— whereby -L₂-O—O—R₂ is formed,    -   X₂ is S or O,    -   X₃ is —CO—O— whereby -L₁CO—O—R₁ is formed,    -   L₃ is a linear or branched alkylene of 1 to 6 carbons,    -   R₃ is a linear or branched alkylene of 1 to 6 carbons, and    -   R₄ and R₅ are the same or different, each hydrogen or a linear        or branched alkyl of 1 to 6 carbons;        or a pharmaceutically acceptable salt thereof.

What is also described herein is the structure

What is also described herein is the compound of formula 2,

wherein

-   -   R₁ and R₂ are the same or different, each a linear or branched        alkyl of 1 to 9 carbons, an alkenyl or alkynyl of 2 to 11        carbons, or cholesteryl,    -   L₁ and L₂ are the same or different, each a linear alkylene or        alkenylene of 5 to 18 carbons,    -   X₁ is —CO—O— whereby -L₂-CO—O—R₂ is formed,    -   X₂ is S or O,    -   X₃ is —CO—O— whereby -L₁-CO—O—R₁ is formed,    -   L₃ is a linear or branched alkylene of 1 to 6 carbons,    -   R₃ is a linear or branched alkylene of 1 to 6 carbons, and    -   R₄ and R₅ are the same or different, each a hydrogen, or a        linear or branched alkyl of 1 to 6 carbons;        or a pharmaceutically acceptable salt thereof.

What is also described herein is the compound of formula 2, in which L₃is methylene; R₁ and R₂ each consists of an alkenyl of at least ninecarbon atoms, R₃ is ethylene or n-propylene, R₄ and R₅ are methyl orethyl, and L₁ and L₂ each consists of a linear alkyl having at leastfive carbon atoms.

What is also described herein is the compound of formula 2, in which L₃is methylene, R₁ and R₂ are the same, an alkyl of 1 or 3 carbons or analkenyl of 9 or 11 carbons; R₃ is ethylene or propylene; R₄ and R₅ aremethyl or ethyl; and/or L₁ and L₂ are the same, an alkylene of 3 or 7carbons, or an alkenylene of 13 or 15 carbons.

What is also described herein is a compound having the structure

What is also described herein is a compound having the structure

What is also described herein is a compound having the structure

What is also described herein is a compound having the structure

What is also described herein is a compound having the structure

What is also described herein is a compound having the structure

What is also described herein is a compound having the structure

What is also described herein is a compound having the structure

What is also described herein is a compound having the structure

What is also described herein is a compound having the structure

What is also described herein is a compound having the structure

What are also described herein are any of the compounds listed inATX-001 to ATX-032 listed in Table 1, below, or a pharmaceuticallyacceptable salt thereof, in a lipid composition, comprising ananoparticle or a bilayer of lipid molecules. The lipid bilayerpreferably further comprises a neutral lipid or a polymer. The lipidcomposition preferably comprises a liquid medium. The compositionpreferably further encapsulates a nucleic acid. The nucleic acidpreferably has an activity of suppressing the expression of the targetgene by utilizing RNA interference (RNAi). The lipid compositionpreferably further comprises a nucleic acid and a neutral lipid or apolymer. The lipid composition preferably encapsulates the nucleic acid.

What is also described herein is a method for introducing a nucleic acidinto a cell of a mammal by using any of the compositions, above. Thecell may be in a liver, lung, kidney, brain, blood, spleen, or bone. Thecomposition preferably is administered intravenously, subcutaneously,intraperitoneally, or intrathecally. The nucleic acid preferably has anactivity of suppressing the expression of a target gene. The target genepreferably is a gene associated with inflammation.

Preferably, the compositions described herein are used in a method fortreating cancer or inflammatory disease. The disease may be one selectedfrom the group consisting of immune disorder, cancer, renal disease,fibrotic disease, genetic abnormality, inflammation, and cardiovasculardisorder.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the knockdown activity of siRNA encapsulated by differentcationic lipids. The lipids include MC3 (0.3 mg/kg), NCl (0.3 mg/kg),ATX-547 (0.3 mg/kg), ATX-001 (0.3 and 1.0 mg/kg), ATX-002 (0.3 and 1.0mg/kg), and ATX-003 (0.3 and 1.0 mg/kg). The amount of Factor VIIknockdown in mouse plasma is shown following administration of the siRNAformulation to C57BL6 mice, compared to injection of vehicle alone. Theamount of Factor VII in abnormal and normal human plasma is included asa control. Statistically significant decreases in Factor VII levels(p<0.01) is shown by an asterix (*).

FIG. 2 shows an evaluation of the effect of siRNA of Factor VII activitybased on the results shown in FIG. 2, and normalized to percentageknockdown compared to the vehicle alone.

FIG. 3 shows the knockdown activity of siRNA encapsulated by differentcationic lipids. The lipids include MC3 (0.3 and 1.5 mg/kg), NCl (0.3mg/kg), AT547 (0.1 and 0.3 mg/kg), AT004 (0.3), AT006 (0.3 and 1.0mg/kg), ATX-010 (0.3 mg/kg), and AT001 (0.3 and 1.5 mg/kg). The amountof Factor VII knockdown in mouse plasma is shown followingadministration of the siRNA formulation to C57BL6 mice, compared toinjection of vehicle alone. The amount of Factor VII in abnormal andnormal human plasma is included as a control. Statistically significantdecreases in Factor VII levels (p<0.01) is shown by an asterix (*).

FIG. 4 shows an evaluation of the effect of siRNA of Factor VII activitybased on the results shown in FIG. 2, and normalized to percentageknockdown compared to the vehicle alone.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

“At least one” means one or more (e.g., 1-3, 1-2, or 1).

“Composition” includes a product comprising the specified ingredients inthe specified amounts, as well as any product that results, directly orindirectly, from combination of the specified ingredients in thespecified amounts.

“In combination with” as used to describe the administration of acompound of formula (1) with other medicaments in the methods oftreatment of this invention, means-that the compounds of formula (1) andthe other medicaments are administered sequentially or concurrently inseparate dosage forms, or are administered concurrently in the samedosage form.

“Mammal” means a human or other mammal, or means a human being.

“Patient” includes both human and other mammals, preferably human.

“Alkyl” is a saturated or unsaturated, straight or branched, hydrocarbonchain. In various embodiments, the alkyl group has 1-18 carbon atoms,i.e. is a C₁-C₁₈ group, or is a C₁-C₁₂ group, a C₁-C₆ group, or a C₁-C₄group. Independently, in various embodiments, the alkyl group has zerobranches (i.e., is a straight chain), one branch, two branches, or morethan two branches. “Alkenyl” is an unsaturated alkyl that may have onedouble bond, two double bonds, more than two double bonds. “Alkynal” isan unsaturated alkyl that may have one triple bond, two triple bonds, ormore than two triple bonds. Alkyl chains may be optionally substitutedwith 1 substituent (i.e., the alkyl group is mono-substituted), or 1-2substituents, or 1-3 substituents, or 1-4 substituents, etc. Thesubstituents may be selected from the group consisting of hydroxy,amino, alkylamino, boronyl, carboxy, nitro, cyano, and the like. Whenthe alkyl group incorporates one or more heteroatoms, the alkyl group isreferred to herein as a heteroalkyl group. When the substituents on analkyl group are hydrocarbons, then the resulting group is simplyreferred to as a substituted alkyl. In various aspects, the alkyl groupincluding substituents has less than 25, 24, 23, 22, 21, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, or 7 carbons.

“Lower alkyl” means a group having about one to about six carbon atomsin the chain which chain may be straight or branched. Non-limitingexamples of suitable alkyl groups include methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, n-pentyl, and hexyl.

“Alkoxy” means an alkyl-O-group wherein alkyl is as defined above.Non-limiting examples of alkoxy groups include: methoxy, ethoxy,n-propoxy, isopropoxy, n-butoxy and heptoxy. The bond to the parentmoiety is through the ether oxygen.

“Alkoxyalkyl” means an alkoxy-alkyl-group in which the alkoxy and alkylare as previously described. Preferred alkoxyalkyl comprise a loweralkyl group. The bond to the parent moiety is through the alkyl.

“Alkylaryl” means an alkyl-aryl-group in which the alkyl and aryl are aspreviously described. Preferred alkylaryls comprise a lower alkyl group.The bond to the parent moiety is through the aryl.

“Aminoalkyl” means an NH₂-alkyl-group, wherein alkyl is as definedabove, bound to the parent moiety through the alkyl group.

“Carboxyalkyl” means an HOOC-alkyl-group, wherein alkyl is as definedabove, bound to the parent moiety through the alkyl group.

“Commercially available chemicals” and the chemicals used in theExamples set forth herein may be obtained from standard commercialsources, where such sources include, for example, Acros Organics(Pittsburgh, Pa.), Sigma-Adrich Chemical (Milwaukee, Wis.), AvocadoResearch (Lancashire, U.K.), Bionet (Cornwall, U.K.), Boron Molecular(Research Triangle Park, N.C.), Combi-Blocks (San Diego, Calif.),Eastman Organic Chemicals, Eastman Kodak Company (Rochester, N.Y.),Fisher Scientific Co. (Pittsburgh, Pa.), Frontier Scientific (Logan,Utah), ICN Biomedicals, Inc. (Costa Mesa, Calif.), Lancaster Synthesis(Windham, N.H.), Maybridge Chemical Co. (Cornwall, U.K.), PierceChemical Co. (Rockford, Ill.), Riedel de Haen (Hannover, Germany),Spectrum Quality Product, Inc. (New Brunswick, N.J.), TCI America(Portland, Oreg.), and Wako Chemicals USA, Inc. (Richmond, Va.).

“Compounds described in the chemical literature” may be identifiedthrough reference books and databases directed to chemical compounds andchemical reactions, as known to one of ordinary skill in the art.Suitable reference books and treatise that detail the synthesis ofreactants useful in the preparation of compounds disclosed herein, orprovide references to articles that describe the preparation ofcompounds disclosed herein, include for example, “Synthetic OrganicChemistry”, John Wiley and Sons, Inc. New York; S. R. Sandler et al,“Organic Functional Group Preparations,” 2^(nd) Ed., Academic Press, NewYork, 1983; H. O. House, “Modern Synthetic Reactions,” 2^(nd) Ed., W. A.Benjamin, Inc. Menlo Park, Calif, 1972; T. L. Glichrist, “HeterocyclicChemistry,” 2^(nd) Ed. John Wiley and Sons, New York, 1992; J. March,“Advanced Organic Chemistry: reactions, Mechanisms and Structure,”5^(th) Ed., Wiley Interscience, New York, 2001; Specific and analogousreactants may also be identified through the indices of known chemicalsprepared by the Chemical Abstract Service of the American ChemicalSociety, which are available in most public and university libraries, aswell as through online databases (the American Chemical Society,Washington, D.C. may be contacted for more details). Chemicals that areknown but not commercially available in catalogs may be prepared bycustom chemical synthesis houses, where many of the standard chemicalsupply houses (such as, those listed above) provide custom synthesisservices.

“Halo” means fluoro, chloro, bromo, or iodo groups. Preferred arefluoro, chloro or bromo, and more preferred are fluoro and chloro.

“Halogen” means fluorine, chlorine, bromine, or iodine. Preferred arefluorine, chlorine and bromine.

“Heteroalkyl” is a saturated or unsaturated, straight or branched, chaincontaining carbon and at least one heteroatom. The heteroalkyl groupmay, in various embodiments, have on heteroatom, or 1-2 heteroatoms, or1-3 heteroatoms, or 1-4 heteroatoms. In one aspect the heteroalkyl chaincontains from 1 to 18 (i.e., 1-18) member atoms (carbon andheteroatoms), and in various embodiments contain 1-12, or 1-6, or 1-4member atoms. Independently, in various embodiments, the heteroalkylgroup has zero branches (i.e., is a straight chain), one branch, twobranches, or more than two branches. Independently, in one embodiment,the hetereoalkyl group is saturated. In another embodiment, theheteroalkyl group is unsaturated. In various embodiments, theunsaturated heterolkyl may have one double bond, two double bonds, morethan two double bonds, and/or one triple bond, two triple bonds, or morethan two triple bonds. Heteroalkyl chains may be substituted orunsubstituted. In one embodiment, the heteroalkyl chain isunsubstituted. In another embodiment, the heteroalkyl chain issubstituted. A substituted heteroalkyl chain may have 1 substituent(i.e., by monosubstituted), or may have 1-2 substituents, or 1-3substituents, or 1-4 substituents, etc. Exemplary heteroalkylsubstituents include esters (—C(O)—O—R) and carbonyls (—C(O)—).

“Hydroxyalkyl” means an HO-alkyl-group, in which alkyl is previouslydefined. Preferred hydroxyalkyls contain lower alkyl. Non-limitingexamples of suitable hydroxyalkyl groups include hydroxymethyl and2-hydroxyethyl.

“Hydrate” is a solvate wherein the solvent molecule is H₂O.

“Solvate” means a physical association of a compound of this disclosurewith one or more solvent molecules. This physical association involvesvarying degrees of ionic and covalent bonding, including hydrogenbonding. In certain instances the solvate will be capable of isolation,for example when one or more solvent molecules are incorporated in thecrystal lattice of the crystalline solid. “Solvate” encompasses bothsolution-phase and isolatable solvates. Non-limiting examples ofsuitable solvates include ethanolates, methanolates, and the like.

The term “substituted” means substitution with specified groups otherthan hydrogen, or with one or more groups, moieties, or radicals whichcan be the same or different, with each, for example, beingindependently selected.

By “antisense nucleic acid”, it is meant a non-enzymatic nucleic acidmolecule that binds to target RNA by means of RNA-RNA or RNA-DNA orRNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566)interactions and alters the activity of the target RNA (for a review,see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al., U.S. Pat.No. 5,849,902). Typically, antisense molecules are complementary to atarget sequence along a single contiguous sequence of the antisensemolecule. However, in certain embodiments, an antisense molecule canbind to substrate such that the substrate molecule forms a loop, and/oran antisense molecule can bind such that the antisense molecule forms aloop. Thus, the antisense molecule can be complementary to two (or evenmore) non-contiguous substrate sequences or two (or even more)non-contiguous sequence portions of an antisense molecule can becomplementary to a target sequence or both. In addition, antisense DNAcan be used to target RNA by means of DNA-RNA interactions, therebyactivating RNase H, which digests the target RNA in the duplex. Theantisense oligonucleotides can comprise one or more RNAse H activatingregion, which is capable of activating RNAse H cleavage of a target RNA.Antisense DNA can be synthesized chemically or expressed via the use ofa single stranded DNA expression vector or equivalent thereof “AntisenseRNA” is an RNA strand having a sequence complementary to a target genemRNA, that can induce RNAi by binding to the target gene mRNA.“Antisense RNA” is an RNA strand having a sequence complementary to atarget gene mRNA, and thought to induce RNAi by binding to the targetgene mRNA. “Sense RNA” has a sequence complementary to the antisenseRNA, and annealed to its complementary antisense RNA to form iNA. Theseantisense and sense RNAs have been conventionally synthesized with anRNA synthesizer.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2′-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

By “RNA” is meant a molecule comprising at least one ribonucleotideresidue. By “ribonucleotide” is meant a nucleotide with a hydroxyl groupat the 2′ position of a β-D-ribo-furanose moiety. The terms includedouble-stranded RNA, single-stranded RNA, isolated RNA such as partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA, as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution and/or alterationof one or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of an interfering RNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA. As used herein, the terms “ribonucleic acid”and “RNA” refer to a molecule containing at least one ribonucleotideresidue, including siRNA, antisense RNA, single stranded RNA, microRNA,mRNA, noncoding RNA, and multivalent RNA. A ribonucleotide is anucleotide with a hydroxyl group at the 2′ position of aβ-D-ribo-furanose moiety. These terms include double-stranded RNA,single-stranded RNA, isolated RNA such as partially purified RNA,essentially pure RNA, synthetic RNA, recombinantly produced RNA, as wellas modified and altered RNA that differs from naturally occurring RNA bythe addition, deletion, substitution, modification, and/or alteration ofone or more nucleotides. Alterations of an RNA can include addition ofnon-nucleotide material, such as to the end(s) of an interfering RNA orinternally, for example at one or more nucleotides of an RNA nucleotidesin an RNA molecule include non-standard nucleotides, such asnon-naturally occurring nucleotides or chemically synthesizednucleotides or deoxynucleotides. These altered RNAs can be referred toas analogs.

By “nucleotide” as used herein is as recognized in the art to includenatural bases (standard), and modified bases well known in the art. Suchbases are generally located at the 1′ position of a nucleotide sugarmoiety. Nucleotides generally comprise a base, sugar, and a phosphategroup. The nucleotides can be unmodified or modified at the sugar,phosphate, and/or base moiety, (also referred to interchangeably asnucleotide analogs, modified nucleotides, non-natural nucleotides,non-standard nucleotides and other; see, for example, Usman andMcSwiggen, supra; Eckstein, et al., International PCT Publication No. WO92/07065; Usman, et al, International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra, all are hereby incorporated by referenceherein). There are several examples of modified nucleic acid bases knownin the art as summarized by Limbach, et al, Nucleic Acids Res. 22:2183,1994. Some of the non-limiting examples of base modifications that canbe introduced into nucleic acid molecules include, inosine, purine,pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidinesor 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others(Burgin, et al., Biochemistry 35:14090, 1996; Uhlman & Peyman, supra).By “modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine, and uracil at 1′ position or theirequivalents.

As used herein complementary nucleotide bases are a pair of nucleotidebases that form hydrogen bonds with each other. Adenine (A) pairs withthymine (T) or with uracil (U) in RNA, and guanine (G) pairs withcytosine (C). Complementary segments or strands of nucleic acid thathybridize (join by hydrogen bonding) with each other. By “complementary”is meant that a nucleic acid can form hydrogen bond(s) with anothernucleic acid sequence either by traditional Watson-Crick or by othernon-traditional modes of binding.

MicroRNAs (miRNA) are single-stranded RNA molecules of about 21-23nucleotides in length, which regulate gene expression miRNAs are encodedby genes that are transcribed from DNA but not translated into protein(non-coding RNA); instead they are processed from primary transcriptsknown as pri-miRNA to short stem-loop structures called pre-miRNA andfinally to functional miRNA. Mature miRNA molecules are partiallycomplementary to one or more messenger RNA (mRNA) molecules, and theirmain function is to downregulate gene expression

As used herein the term small interfering RNA (siRNA), sometimes knownas short interfering RNA or silencing RNA, is used to refer to a classof double-stranded RNA molecules, 16-40 nucleotides in length, that playa variety of roles in biology. Most notably, siRNA is involved in theRNA interference (RNAi) pathway, where it interferes with the expressionof a specific gene. In addition to their role in the RNAi pathway,siRNAs also act in RNAi-related pathways, e.g., as an antiviralmechanism or in shaping the chromatin structure of a genome; thecomplexity of these pathways is only now being elucidated.

As used herein, the term RNAi refers to an RNA-dependent gene silencingprocess that is controlled by the RNA-induced silencing complex (RISC)and is initiated by short double-stranded RNA molecules in a cell, wherethey interact with the catalytic RISC component argonaute. When thedouble-stranded RNA or RNA-like iNA or siRNA is exogenous (coming frominfection by a virus with an RNA genome or from transfected iNA orsiRNA), the RNA or iNA is imported directly into the cytoplasm andcleaved to short fragments by the enzyme dicer. The initiating dsRNA canalso be endogenous (originating in the cell), as in pre-microRNAsexpressed from RNA-coding genes in the genome. The primary transcriptsfrom such genes are first processed to form the characteristic stem-loopstructure of pre-miRNA in the nucleus, then exported to the cytoplasm tobe cleaved by dicer. Thus, the two dsRNA pathways, exogenous andendogenous, converge at the RISC complex. The active components of anRNA-induced silencing complex (RISC) are endonucleases called argonauteproteins, which cleave the target mRNA strand complementary to theirbound siRNA or iNA. As the fragments produced by dicer aredouble-stranded, they could each in theory produce a functional siRNA oriNA. However, only one of the two strands, which is known as the guidestrand, binds the argonaute protein and directs gene silencing. Theother anti-guide strand or passenger strand is degraded during RISCactivation.

The compounds of formula (1) form salts that are also within the scopeof this disclosure. Reference to a compound of formula (1) herein isunderstood to include reference to salts thereof, unless otherwiseindicated. The term “salt(s)”, as employed herein, denotes acidic saltsformed with inorganic and/or organic acids, as well as basic saltsformed with inorganic and/or organic bases. In addition, when a compoundof formula (1) contains both a basic moiety, such as, but not limitedto, a pyridine or imidazole, and an acidic moiety, such as, but notlimited to, a carboxylic acid, zwitterions (“inner salts”) may be formedand are included within the term “salt(s)” as used herein. The salts canbe pharmaceutically acceptable (i.e., non-toxic, physiologicallyacceptable) salts, although other salts are also useful. Salts of thecompounds of the formula (1) may be formed, for example, by reacting acompound of formula (1) with an amount of acid or base, such as anequivalent amount, in a medium such as one in which the saltprecipitates or in an aqueous medium followed by lyophilization.

Exemplary acid addition salts include acetates, adipates, alginates,ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates,borates, butyrates, citrates, camphorates, camphorsulfonates,cyclopentanepropionates, digluconates, dodecylsulfates,ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates,hemisulfates, heptanoates, hexanoates, hydrochlorides, hydrobromides,hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates,methanesulfonates, 2-napthalenesulfonates, nicotinates, nitrates,oxalates, pectinates, persulfates, 3-phenylpropionates, phosphates,picrates, pivalates, propionates, salicylates, succinates, sulfates,sulfonates (such as those mentioned herein), tartarates, thiocyanates,toluenesulfonates (also known as tosylates) undecanoates, and the like.Additionally, acids which are generally considered suitable for theformation of pharmaceutically useful salts from basic pharmaceuticalcompounds are discussed, for example, by S. Berge et al, J.Pharmaceutical Sciences (1977) 66(1)1-19; P. Gould, International J.Pharmaceutics (1986) 33 201-217; Anderson et al, The Practice ofMedicinal Chemistry (1996), Academic Press, New York; and in The OrangeBook (Food & Drug Administration, Washington, D.C. on their website).These disclosures are incorporated by reference herein.

Exemplary basic salts include ammonium salts, alkali metal salts such assodium, lithium, and potassium salts, alkaline earth metal salts such ascalcium and magnesium salts, salts with organic bases (for example,organic amines) such as benzathines, dicyclohexylamines, hydrabamines(formed with N,N-bis(dehydroabietyl)ethylenediamine),N-methyl-D-glucamines, N-methyl-D-glucamides, t-butyl amines, and saltswith amino acids such as arginine, lysine, and the like. Basicnitrogen-containing groups may be quarternized with agents such as loweralkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides,bromides, and iodides), dialkyl sulfates (e.g., dimethyl, diethyl,dibutyl, and diamyl sulfates), long chain halides (e.g., decyl, lauryl,myristyl, and stearyl chlorides, bromides, and iodides), arylalkylhalides (e.g., benzyl and phenethyl bromides), and others.

All such acid and base salts are intended to be pharmaceuticallyacceptable salts within the scope of the disclosure and all acid andbase salts are considered equivalent to the free forms of thecorresponding compounds for purposes of the disclosure.

Compounds of formula (1) can exist in unsolvated and solvated forms,including hydrated forms. In general, the solvated forms, withpharmaceutically acceptable solvents such as water, ethanol, and thelike, are equivalent to the unsolvated forms for the purposes of thisdisclosure.

Compounds of formula (1) and salts, solvates thereof, may exist in theirtautomeric form (for example, as an amide or imino ether). All suchtautomeric forms are contemplated herein as part of the presentdisclosure.

Also within the scope of the present disclosure are polymorphs of thecompounds of this disclosure (i.e., polymorphs of the compounds offormula 1 are within the scope of this disclosure).

All stereoisomers (for example, geometric isomers, optical isomers, andthe like) of the present compounds (including those of the salts,solvates, and prodrugs of the compounds as well as the salts andsolvates of the prodrugs), such as those which may exist due toasymmetric carbons on various substituents, including enantiomeric forms(which may exist even in the absence of asymmetric carbons), rotamericforms, atropisomers, and diastereomeric forms, are contemplated withinthe scope of this disclosure. Individual stereoisomers of the compoundsof this disclosure may, for example, be substantially free of otherisomers, or may be admixed, for example, as racemates or with all other,or other selected, stereoisomers. The chiral centers of the compoundsherein can have the S or R configuration as defined by the IUPAC 1974Recommendations. The use of the terms “salt”, “solvate”, and the like,is intended to equally apply to the salt and solvate of enantiomers,stereoisomers, rotamers, tautomers, racemates, or prodrugs of thedisclosed compounds.

Classes of compounds that can be used as the chemotherapeutic agent(antineoplastic agent) include: alkylating agents, antimetabolites,natural products and their derivatives, hormones and steroids (includingsynthetic analogs), and synthetics. Examples of compounds within theseclasses are given below.

Cationic Lipids

The description includes synthesis of certain cationic lipid compounds.The compounds are particularly suitable for delivering polynucleotidesto cells and tissues as demonstrated in subsequent sections. Thelipomacrocycle compound described herein may be used for other purposesas well as, for example, recipients and additives.

The synthetic methods for the cationic lipid compounds can besynthesized with the skills in the art. The skilled of the art willrecognize other methods to produce these compounds, and to produce alsothe other compounds of the description.

The cationic lipid compounds may be combined with an agent to formmicroparticles, nanoparticles, liposomes, or micelles. The agent to bedelivered by the particles, liposomes, or micelles may be in the form ofa gas, liquid, or solid, and the agent may be a polynucleotide, protein,peptide, or small molecule. The lipomacrocycle compounds may be combinedwith other cationic lipid compounds, polymers (synthetic or natural),surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to formthe particles. These particles may then optionally be combined with apharmaceutical excipient to form a pharmaceutical composition.

The present description provides novel cationic lipid compounds and drugdelivery systems based on the use of such cationic lipid compounds. Thesystem may be used in the pharmaceutical/drug delivery arts to deliverpolynucleotides, proteins, small molecules, peptides, antigen, drugs,etc. to a patient, tissue, organ, or cell. These novel compounds mayalso be used as materials for coating, additives, excipients, materials,or bioengineering.

The cationic lipid compounds of the present description provide forseveral different uses in the drug delivery art. The amine-containingportion of the cationic lipid compounds may be used to complexpolynucleotides, thereby enhancing the delivery of polynucleotide andpreventing their degradation. The cationic lipid compounds may also beused in the formation of picoparticles, nanoparticles, microparticles,liposomes, and micelles containing the agent to be delivered.Preferably, the cationic lipid compounds are biocompatible andbiodegradable, and the formed particles are also biodegradable andbiocompatible and may be used to provide controlled, sustained releaseof the agent to be delivered. These and their corresponding particlesmay also be responsive to pH changes given that these are protonated atlower pH. They may also act as proton sponges in the delivery of anagent to a cell to cause endosome lysis.

In certain embodiments, the cationic lipid compounds are relativelynon-cytotoxic. The cationic lipid compounds may be biocompatible andbiodegradable. The cationic lipid may have pK_(a) s in the range ofapproximately 5.5 to approximately 7.5, more preferably betweenapproximately 6.0 and approximately 7.0. It may be designed to have adesired pK_(a) between approximately 3.0 and approximately 9.0, orbetween approximately 5.0 and approximately 8.0. The cationic lipidcompounds described herein are particularly attractive for drug deliveryfor several reasons: they contain amino groups for interacting with DNA,RNA, other polynucleotides, and other negatively charged agents, forbuffering the pH, for causing endo-osmolysis, for protecting the agentto be delivered, they can be synthesized from commercially availablestarting materials; and/or they are pH responsive and can be engineeredwith a desired pK_(a).

A composition containing a cationic lipid compound may be 30-70%cationic lipid compound, 0-60% cholesterol, 0-30% phospholipid and 1-10%polyethylene glycol (PEG). Preferably, the composition is 30-40%cationic lipid compound, 40-50% cholesterol, and 10-20% PEG. In otherpreferred embodiments, the composition is 50-75% cationic lipidcompound, 20-40% cholesterol, and 5 to 10% phospholipid, and 1-10% PEG.The composition may contain 60-70% cationic lipid compound, 25-35%cholesterol, and 5-10% PEG. The composition may contain up to 90%cationic lipid compound and 2 to 15% helper lipid.

The formulation may be a lipid particle formulation, for examplecontaining 8-30% compound, 5-30% helper lipid, and 0-20% cholesterol;4-25% cationic lipid, 4-25% helper lipid, 2 to 25% cholesterol, 10 to35% cholesterol-PEG, and 5% cholesterol-amine; or 2-30% cationic lipid,2-30% helper lipid, 1 to 15% cholesterol, 2 to 35% cholesterol-PEG, and1-20% cholesterol-amine; or up to 90% cationic lipid and 2-10% helperlipids, or even 100% cationic lipid.

Compositions and Formulations for Administration

The nucleic acid-lipid compositions of this disclosure may beadministered by various routes, for example, to effect systemic deliveryvia intravenous, parenteral, intraperitoneal, or topical routes. In someembodiments, a siRNA may be delivered intracellularly, for example, incells of a target tissue such as lung or liver, or in inflamed tissues.In some embodiments, this disclosure provides a method for delivery ofsiRNA in vivo. A nucleic acid-lipid composition may be administeredintravenously, subcutaneously, or intraperitoneally to a subject. Insome embodiments, the disclosure provides methods for in vivo deliveryof interfering RNA to the lung of a mammalian subject.

In some embodiments, this disclosure provides a method of treating adisease or disorder in a mammalian subject. A therapeutically effectiveamount of a composition of this disclosure containing a nucleic, acationic lipid, an amphiphile, a phospholipid, cholesterol, and aPEG-linked cholesterol may be administered to a subject having a diseaseor disorder associated with expression or overexpression of a gene thatcan be reduced, decreased, downregulated, or silenced by thecomposition.

The compositions and methods of the disclosure may be administered tosubjects by a variety of mucosal administration modes, including byoral, rectal, vaginal, intranasal, intrapulmonary, or transdermal ordermal delivery, or by topical delivery to the eyes, ears, skin, orother mucosal surfaces. In some aspects of this disclosure, the mucosaltissue layer includes an epithelial cell layer. The epithelial cell canbe pulmonary, tracheal, bronchial, alveolar, nasal, buccal, epidermal,or gastrointestinal. Compositions of this disclosure can be administeredusing conventional actuators such as mechanical spray devices, as wellas pressurized, electrically activated, or other types of actuators.

Compositions of this disclosure may be administered in an aqueoussolution as a nasal or pulmonary spray and may be dispensed in sprayform by a variety of methods known to those skilled in the art.Pulmonary delivery of a composition of this disclosure is achieved byadministering the composition in the form of drops, particles, or spray,which can be, for example, aerosolized, atomized, or nebulized.Particles of the composition, spray, or aerosol can be in either aliquid or solid form. Preferred systems for dispensing liquids as anasal spray are disclosed in U.S. Pat. No. 4,511,069. Such formulationsmay be conveniently prepared by dissolving compositions according to thepresent disclosure in water to produce an aqueous solution, andrendering said solution sterile. The formulations may be presented inmulti-dose containers, for example in the sealed dispensing systemdisclosed in U.S. Pat. No. 4,511,069. Other suitable nasal spraydelivery systems have been described in Transdermal Systemic Medication,Y. W. Chien ed., Elsevier Publishers, New York, 1985; and in U.S. Pat.No. 4,778,810. Additional aerosol delivery forms may include, e.g.,compressed air-, jet-, ultrasonic-, and piezoelectric nebulizers, whichdeliver the biologically active agent dissolved or suspended in apharmaceutical solvent, e.g., water, ethanol, or mixtures thereof.

Nasal and pulmonary spray solutions of the present disclosure typicallycomprise the drug or drug to be delivered, optionally formulated with asurface active agent, such as a nonionic surfactant (e.g.,polysorbate-80), and one or more buffers. In some embodiments of thepresent disclosure, the nasal spray solution further comprises apropellant. The pH of the nasal spray solution may be from about pH 6.8to 7.2. The pharmaceutical solvents employed can also be a slightlyacidic aqueous buffer of pH 4-6. Other components may be added toenhance or maintain chemical stability, including preservatives,surfactants, dispersants, or gases.

In some embodiments, this disclosure is a pharmaceutical product whichincludes a solution containing a composition of this disclosure and anactuator for a pulmonary, mucosal, or intranasal spray or aerosol.

A dosage form of the composition of this disclosure can be liquid, inthe form of droplets or an emulsion, or in the form of an aerosol.

A dosage form of the composition of this disclosure can be solid, whichcan be reconstituted in a liquid prior to administration. The solid canbe administered as a powder. The solid can be in the form of a capsule,tablet, or gel.

To formulate compositions for pulmonary delivery within the presentdisclosure, the biologically active agent can be combined with variouspharmaceutically acceptable additives, as well as a base or carrier fordispersion of the active agent(s). Examples of additives include pHcontrol agents such as arginine, sodium hydroxide, glycine, hydrochloricacid, citric acid, and mixtures thereof. Other additives include localanesthetics (e.g., benzyl alcohol), isotonizing agents (e.g., sodiumchloride, mannitol, sorbitol), adsorption inhibitors (e.g., Tween 80),solubility enhancing agents (e.g., cyclodextrins and derivativesthereof), stabilizers (e.g., serum albumin), and reducing agents (e.g.,glutathione). When the composition for mucosal delivery is a liquid, thetonicity of the formulation, as measured with reference to the tonicityof 0.9% (w/v) physiological saline solution taken as unity, is typicallyadjusted to a value at which no substantial, irreversible tissue damagewill be induced in the mucosa at the site of administration. Generally,the tonicity of the solution is adjusted to a value of about 1/3 to 3,more typically 1/2 to 2, and most often 3/4 to 1.7.

The biologically active agent may be dispersed in a base or vehicle,which may comprise a hydrophilic compound having a capacity to dispersethe active agent and any desired additives. The base may be selectedfrom a wide range of suitable carriers, including but not limited to,copolymers of polycarboxylic acids or salts thereof, carboxylicanhydrides (e.g. maleic anhydride) with other monomers (e.g.,methyl(meth)acrylate, acrylic acid, etc.), hydrophilic vinyl polymerssuch as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone,cellulose derivatives such as hydroxymethylcellulose,hydroxypropylcellulose, etc., and natural polymers such as chitosan,collagen, sodium alginate, gelatin, hyaluronic acid, and nontoxic metalsalts thereof. Often, a biodegradable polymer is selected as a base orcarrier, for example, polylactic acid, poly(lactic acid-glycolic acid)copolymer, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolicacid) copolymer and mixtures thereof. Alternatively or additionally,synthetic fatty acid esters such as polyglycerin fatty acid esters,sucrose fatty acid esters, etc., can be employed as carriers.Hydrophilic polymers and other carriers can be used alone or incombination, and enhanced structural integrity can be imparted to thecarrier by partial crystallization, ionic bonding, crosslinking, and thelike. The carrier can be provided in a variety of forms, including fluidor viscous solutions, gels, pastes, powders, microspheres, and films fordirect application to the nasal mucosa. The use of a selected carrier inthis context may result in promotion of absorption of the biologicallyactive agent.

Formulations for mucosal, nasal, or pulmonary delivery may contain ahydrophilic low molecular weight compound as a base or excipient. Suchhydrophilic low molecular weight compounds provide a passage mediumthrough which a water-soluble active agent, such as a physiologicallyactive peptide or protein, may diffuse through the base to the bodysurface where the active agent is absorbed. The hydrophilic lowmolecular weight compound optionally absorbs moisture from the mucosa orthe administration atmosphere and dissolves the water-soluble activepeptide. The molecular weight of the hydrophilic low molecular weightcompound is generally not more than 10,000 and preferably not more than3000. Examples of hydrophilic low molecular weight compounds includepolyol compounds, such as oligo-, di- and monosaccarides includingsucrose, mannitol, lactose, L-arabinose, D-erythrose, D-ribose,D-xylose, D-mannose, D-galactose, lactulose, cellobiose, gentibiose,glycerin, polyethylene glycol, and mixtures thereof. Further examples ofhydrophilic low molecular weight compounds include N-methylpyrrolidone,alcohols (e.g., oligovinyl alcohol, ethanol, ethylene glycol, propyleneglycol, etc.), and mixtures thereof.

The compositions of this disclosure may alternatively contain aspharmaceutically acceptable carriers substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, and wetting agents, for example,sodium acetate, sodium lactate, sodium chloride, potassium chloride,calcium chloride, sorbitan monolaurate, triethanolamine oleate, andmixtures thereof. For solid compositions, conventional nontoxicpharmaceutically acceptable carriers can be used which include, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, talcum, cellulose, glucose, sucrose,magnesium carbonate, and the like.

In certain embodiments of the disclosure, the biologically active agentmay be administered in a time release formulation, for example in acomposition which includes a slow release polymer. The active agent canbe prepared with carriers that will protect against rapid release, forexample a controlled release vehicle such as a polymer,microencapsulated delivery system or bioadhesive gel. Prolonged deliveryof the active agent, in various compositions of the disclosure can bebrought about by including in the composition agents that delayabsorption, for example, aluminum monosterate hydrogels and gelatin.

While this disclosure has been described in relation to certainembodiments, and many details have been set forth for purposes ofillustration, it will be apparent to those skilled in the art that thisdisclosure includes additional embodiments, and that some of the detailsdescribed herein may be varied considerably without departing from thisdisclosure. This disclosure includes such additional embodiments,modifications and equivalents. In particular, this disclosure includesany combination of the features, terms, or elements of the variousillustrative components and examples.

EXAMPLES Example 1

Exemplary compounds of formula (1) are provided in Table 1.

TABLE 1 KD @ Lipid 0.3 ID Novel Lipid MW pKa mg/kg ATX- 001

695.1 8.9 ~0 ATX- 002

681 8.7 98 ATX- 003

695.1 9.3 ~0 ATX- 004

709.13 9.4 ~0 ATX- 005

709.13 9.0 ~0 ATX- 006

723.15 9.8 ~0 ATX- 007

723.15 9.5 n/a ATX- 008

737.18 10.3 n/a ATX- 009

695.1 8.8 ~0 ATX- 010

709.13 9.6 30 ATX- 011

709.13 9.4 n/a ATX- 012

723.15 10.2 ~0 ATX- 013

681.01 n/a ATX- 014

695.1 n/a ATX- 015

695.1 n/a ATX- 016

709.13 15 ATX- 017

695.1 n/a ATX- 018

554.92 40 (@ .05 mpk) ATX- 019

611.03 30 (@ .05 mpk) ATX- 020

667.13 40 (@ .05 mpk) ATX- 021

679.04 n/a ATX- 022

665.01 n/a ATX- 023

695.1 n/a ATX- 024

925.5 0 ATX- 025

869.39 15 ATX- 026

681.07 n/a ATX- 027

695.1 n/a ATX- 028

681.07 n/a ATX- 029

681.1 n/a ATX- 030

695.1 n/a ATX- 031

663.1 n/a ATX- 032

645.13 n/a

Table 1 shows the name and structure of each compound, its molecularweight, its pKa, and its knockdown bioactivity (KD) in an assaydescribed below in Example 19.

Example 2 Synthesis of Methyl 8-bromooctanoate

Chemicals/ S. No. Reagents and solvents M. Wt. Moles Eq. Wt. 18-Bromooctanoic acid 223 269.05 1 60 gm. 2 Dry MeOH 400 ml 3 Con H₂SO₄10 drop

Under N2 atmosphere, 8-bromooctanoic acid was dissolved in dry methanol.Concentrated H₂SO₄ was added drop-wise and the reaction mixture wasstirred under reflux for three hours.

The reaction was monitored by thin layer chromatography until completed.Solvent was completely removed under vacuum. The reaction mixture wasdiluted with ethyl acetate and washed with water. The water layer wasre-extracted with ethyl acetate. The total organic layer was washed witha saturated NaHCO₃ solution. The organic layer was washed again withwater and finally washed with brine. The product was dried overanhydrous Na₂SO₄ and concentrated.

Example 3 Synthesis of Dimethyl 8,8′-(benzanediyl)dioctanoate

Chemicals/ S. No. Reagents and solvents M. Wt. Moles Eq. Wt. 1 Benzylamine 107 126.54 1 13.54 2 Methyl 8-bromooctanoate 237 253.08 2 60 g 3Dry K₂CO₃ 138 759.25 6 104.7 4 Dry DMF 500 ml

Dry K₂CO₃ was taken and added to dry dimethylformamide under N₂. Benzylamine in dimethylformamide was slowly added. Methyl 8-bromooctanoatedissolved in dimethylformamide was then added at room temperature. Thereaction mixture was heated to 80° C. and the reaction was maintainedfor 36 hours with stirring.

The reaction was monitored by thin layer chromatography until completed.The reaction product was cooled to room temperature and water was added.The compound was extracted with ethyl acetate. The water layer wasre-extracted with ethyl acetate. The total organic layer was washed withwater and finally with brine solution. The product was dried overanhydrous Na₂SO₄ and concentrated.

The reaction product was purified by silica gel column chromatography in3% methanol in chloroform 44 gm of pure product was recovered.

Using TLC system of 10% methanol in chloroform, the product migratedwith a Rf: 0.8, visualizing by charring in ninhydrine. The overall yieldwas 82%. The compound was a light brown liquid. The structure wasconfirmed by ¹H-NMR.

Example 4 Synthesis of Dimethyl 8,8′-azanediyldioctanoate

Chemicals/ S. No. Reagents and solvents M. Wt. mmoles Eq. Wt. 1 Dimethyl8,8′- 419.60 8.34 1 3.5 gm (benzanediyl)dioctanoate 2 10% Pd/C 20% wt700 mg 3 Ethanol 90 ml

Dimethyl 8,8′-(benzanediyOdioctanoate was transferred to hydrogenationglass vessel, and ethanol was added followed by 10% Pd/C. The reactionmixture was shaken in a Parr-shaker apparatus under 50 pounds per squareinch H₂ atmosphere pressure for two hours at room temperature.

The reaction product was filtered through celite and washed with hotethyl acetate. The filtrate was concentrated under vacuum.

Example 5 Synthesis of Dimethyl 8,8′-((tertbutoxycarbonyl)azanedil)dioctanoate

S. No Chemicals/reagents/solvents Mw Mole's Eq wt 1 Dimethyl 8,8′- 3290.0972 1 32 gm azanediyldioctanoate 2 Boc anhydride 218 0.145 1.5 31.3gm 3 Et₃N (Dry) 101 0.389 4 9 gm 4 DCM(Dry) 700 ml

Dimethyl 8,8′-azanediyldioctanoate was transferred to DCM and Et₃N tothe reaction mass and cooled to 0° C. Boc anhydride diluted in DCM wasadded drop to the above reaction. After the addition was completed, thereaction mixture was stirred at room temperature for three hours.

The reaction was quenched with water and the DCM layer was separated.The water phase was re-extracted with DCM and the combined DCM layerswere washed with brine solution and dried with Na₂SO₄. Afterconcentration, 40 gm of crude compound was collected.

Crude reaction product was purified by column chromatography using 0-12%ethyl acetate in hexane. The yield recovered was 48%. A single productmigrated by thin layer chromatography in 20% ethyl acetate in hexanewith an Rf of 0.5, charring with ninhydrine.

Example 6 Synthesis of 8,8′-((tertbutoxycarbonyl)azanediyl) dioctanoicacid

S. No Chemicals/reagents/solvents Mw Mole's Eq wt 1 Dimethyl 8,8′ 4290.0489 1 21 gm ((tertbutoxycarbonyl)azanediyl) dioctanoate 2 6N NaOH(aq.) 175 ml 3 Dry THF 200 ml

Dimethyl 8,8′-((tertbutoxycarbonyl)azanedil) dioctanoate was transferredto THF. A 6N sodium hydroxide solution was added at room temperature.The reaction was maintained with stirring overnight at room temperature.

Reaction mass was evaporated under vacuum at 25° C. to remove THF. Thereaction product was acidified with 5N HCl. Ethyl acetate was added tothe aqueous layer. The separated organic layer was washed with water andthe water layer was re-extracted with ethyl acetate. The combinedorganic layers were washed with brine solution and dried over anhydrousNa₂SO₄. Concentration of the solution gave 18 gm of crude mass.

Example 7 Synthesis of Di((Z)-non-2-en-1-yl)8,8′((tertbutoxycarbonyl)azanediyl)

S. No Chemicals/reagents/solvents Mw Mole's Eq wt 1 8,8^(′)- 549.50.03275 1 18 gm ((tertbutoxy- carbonyl)azanediyl) dioctanoic acid 2Cis-2-nonene-1-ol 142.24 0.065514 2 9.31 gm 3 HATU 380.23 0.06878 2.126.15 gm 4 Di-Isopropyl ethyl amine 129.25 0.1146 3.5 14.81 gm 5 DMAP122.17 0.003275 0.1 400 mg 6 Dry-DCM 150 ml

8,8′-((tertbutoxycarbonyl)azanediyl) dioctanoic acid was dissolved indry DCM. HATU was added to this solution. Di-isopropyl ethyl amine wasadded slowly to the reaction mixture at room temperature. The internaltemp rose to 40° C. and a pale yellow color solution was formed. DMAPwas added to the reaction mixture followed by cis-2-nonene-1-ol solutionin dry DCM. The reaction changed to brown color. The reaction wasstirred for five hours at room temperature.

The reaction was checked by thin layer chromatography under completion.Water was added to the reaction product, which was extracted with DCM.The DCM layer was washed with water followed by brine solution. Theorganic layer was dried over anhydrous Na₂SO₄ and concentrated to obtain35 gm of crude compound.

Example 8 Synthesis of ATX-001

Di((Z)-non-2-en-1-yl) 8,8′((tertbutoxycarbonyl)azanediyl) dioctanoate(0.023 mol, 15 g) was dissolved in dry dichloromethane (DCM) (200 ml).Trifluoroacetic acid (TFA) was added at 0° C. to initiate a reaction.The reaction temperature was slowly allowed to warm to room temperatureover for 30 minutes with stirring. Thin layer chromatography showed thatthe reaction was completed. The reaction product was concentrated undervacuum at 40° C. and the crude residue was diluted with DCM, and washedwith a 10% NaHCO₃ solution. The aqueous layer was re-extracted with DCM,and the combined organic layers were washed with brine solution, driedover Na₂SO₄ and concentrated. The collected crude product (12 grams) wasdissolved in dry DCM (85 ml) under nitrogen gas. Triphosgene were addedand the reaction mixture was cooled to 0° C., and Et₃N was added dropwise. The reaction mixture was stirred overnight at room temperature.Thin layer chromatography showed that the reaction was completed. DCMsolvent was removed from the reaction mass by distillation under N₂. Thereaction product was cooled to 0° C., diluted with DCM (50 ml), and2-((2-(dimethylamino)ethyl)thio) acetic acid (0.039 mol, 6.4g) andcarbodiimide (EDC HCl) (0.054 mol, 10.4 g). The reaction mixture wasthen stirred overnight at room temperature. Thin layer chromatographyshowed that the reaction was completed. The reaction product was dilutedwith 0.3M HCl solution (75 ml), and the organic layer was separated. Theaqueous layer was re-extracted with DCM, and the combined organic layerswere washed with 10% K₂CO₃ aqueous solution (75m1) and dried overanhydrous Na₂SO₄. Concentration of the solvent gave a crude mass of 10gram. The crude compound was purified by silica gel column (100-200mesh) using 3% MeOH/DCM. The yield was 10.5 g (68%).

Example 9 Synthesis of ATX-002

Di((Z)-non-2-en-1-yl) 8,8′((tertbutoxycarbonyl)azanediyl) dioctanoate(13.85 mmol, 9 grams) was dissolved in dry DCM (150 ml). TFA was addedat 0° C. to initiate a reaction. The reaction temperature was slowlyallowed to warm to room temperature over for 30 minutes with stirring.Thin layer chromatography showed that the reaction was completed. Thereaction product was concentrated under vacuum at 40° C. and the cruderesidue was diluted with DCM, and washed with a 10% NaHCO₃ solution. Theaqueous layer was re-extracted with DCM, and the combined organic layerswere washed with brine solution, dried over Na₂SO₄ and concentrated. Thecollected crude product was dissolved in dry DCM (85 ml) under nitrogengas. Triphosgene were added and the reaction mixture was cooled to 0°C., and Et₃N was added drop wise. The reaction mixture was stirredovernight at room temperature. Thin layer chromatography showed that thereaction was completed. DCM solvent was removed from the reaction massby distillation under N₂. The reaction product was cooled to 0° C.,diluted with DCM (50 ml), and 2-(dimethylamino)ethanethiol HCl (0.063mol, 8.3 g) was added, followed by Et₃N (dry). The reaction mixture wasthen stirred overnight at room temperature. Thin layer chromatographyshowed that the reaction was completed. The reaction product was dilutedwith 0.3M HCl solution (75 ml), and the organic layer was separated. Theaqueous layer was re-extracted with DCM, and the combined organic layerswere washed with 10% K₂CO₃ aqueous solution (75 ml) and dried overanhydrous Na₂SO₄. Concentration of the solvent gave a crude mass of 10gram. The crude compound was purified by silica gel column (100-200mesh) using 3% MeOH/DCM. The yield was 3.1 gram.

Example 10 Synthesis of ATX-003

Di((Z)-non-2-en-1-yl) 8,8′((tertbutoxycarbonyl)azanediyl) dioctanoate(0.00337 mol, 2.2 g) was dissolved in dry DCM (20 ml). TFA was added at0° C. to initiate a reaction. The reaction temperature was slowlyallowed to warm to room temperature over for 30 minutes with stirring.Thin layer chromatography showed that the reaction was completed. Thereaction product was concentrated under vacuum at 40° C. and the cruderesidue was diluted with DCM, and washed with a 10% NaHCO₃ solution. Theaqueous layer was re-extracted with DCM, and the combined organic layerswere washed with brine solution, dried over Na₂SO₄ and concentratedunder reduced pressure. The collected crude product was dissolved in dryDCM (10 ml) under nitrogen gas. Triphosgene (0.0182 mol, 5.4 g) wasadded and the reaction mixture was cooled to 0° C., and Et₃N was addeddrop wise. The reaction mixture was stirred overnight at roomtemperature. Thin layer chromatography showed that the reaction wascompleted. DCM solvent was removed from the reaction mass bydistillation under N₂. The reaction product was cooled to 0° C., dilutedwith DCM (15 ml), and 2-(dimethylamino)propanethiol HCl (0.0182 mol,2.82 g) was added, followed by Et₃N (dry). The reaction mixture was thenstirred overnight at room temperature. Thin layer chromatography showedthat the reaction was completed. The reaction product was diluted with0.3 M HCl aqueous solution (20 ml), and the organic layer was separated.The aqueous layer was re-extracted with DCM, and the combined organiclayers were washed with 10% K₂CO₃ aqueous solution (50 ml) and driedover anhydrous Na₂SO₄. Concentration of the solvent gave a crude mass of5 gram. The crude compound was purified by silica gel column (100-200mesh) using 3% MeOH/DCM. The yield was 0.9 gram.

Example 11 Synthesis of ATX-004

Di((Z)-non-2-en-1-yl) 8,8′((tertbutoxycarbonyl)azanediyl) dioctanoate(0.023 mol, 15 g) was dissolved in DCM (200 ml). TFA was added at 0° C.to initiate a reaction. The reaction temperature was slowly allowed towarm to room temperature over for 30 minutes with stirring. Thin layerchromatography showed that the reaction was completed. The reactionproduct was concentrated under vacuum at 40° C. and the crude residuewas diluted with DCM, and washed with a 10% NaHCO₃ solution. The aqueouslayer was re-extracted with DCM, and the combined organic layers werewashed with brine solution, dried over Na₂SO₄ and concentrated. Thecollected crude product, di((Z)-non-2-en-1-yl)8,8′-azanediyldioctanoate(5.853 mmol, 3.2 g) was dissolved in dry dimethyl formamide (DMF) undernitrogen, and 2-((3-(dimethylamino)propyl)thio)acetic acid (10.48 mmol,1.85 g) and EDC HCl (14.56 mmol, 2.78 g) was added. The reaction mixturewas stirred for overnight at room temperature. The reaction was quenchedwith water (30 ml) and diluted with DCM (30 ml), and the organic layerwas separated. The aqueous layer was re-extracted with DCM, and thecombined organic layers were washed with 10% K₂CO₃ aqueous solution anddried over anhydrous Na₂SO₄. The crude compound was purified by silicagel column (100-200 mesh) using 3% MeOH/DCM. The yield was 1 gram(24.2%).

Example 12 Synthesis of ATX-005

Di((Z)-non-2-en-1-yl) 8,8′((tertbutoxycarbonyl)azanediyl) dioctanoate(0.023 mol, 15 g) was dissolved in dry DCM (200 ml). TFA was added at 0°C. to initiate a reaction. The reaction temperature was slowly allowedto warm to room temperature over for 30 minutes with stirring. Thinlayer chromatography showed that the reaction was completed. Thereaction product was concentrated under vacuum at 40° C. and the cruderesidue was diluted with DCM, and washed with a 10% NaHCO₃ solution. Theaqueous layer was re-extracted with DCM, and the combined organic layerswere washed with brine solution, dried over Na₂SO₄ and concentrated.Crude reaction product, di((Z)-non-2-en-1-yl)8,8′-azanediyldioctanoate(5.853 mmol, 3.2 g) was dissolved in DMF under nitrogen gas.2-((3-(dimethylamino)propyl)thio)acetic acid (10.48 mmol, 1.85 g) andEDC HCl (14.56 mmol, 2.78 g) were added and the reaction mixture wasstirred overnight at room temperature. Thin layer chromatography showedthat the reaction was completed. The reaction product was quenched withwater (30 ml) and diluted with DCM (30 ml). The aqueous layer wasre-extracted with DCM, and the combined organic layers were washed with10% K₂CO₃ aqueous solution (75 ml) and dried over anhydrous Na₂SO₄.Concentration of the solvent gave a crude mass of 5 gram. Crude compoundwas purified by silica gel column (100-200 mesh) using 3% MeOH/DCM. Theyield was 1 gram (24.2%).

Example 13 Synthesis of ATX-006

Di((Z)-non-2-en-1-yl) 8,8′((tertbutoxycarbonyl)azanediyl) dioctanoatewas dissolved in dry DCM (150 ml). TFA was added at 0° C. to initiate areaction. The reaction temperature was slowly allowed to warm to roomtemperature over for 30 minutes with stirring. Thin layer chromatographyshowed that the reaction was completed. The reaction product wasconcentrated under vacuum at 40° C. and the crude residue was dilutedwith DCM, and washed with a 10% NaHCO₃ solution. The aqueous layer wasre-extracted with DCM, and the combined organic layers were washed withbrine solution, dried over Na₂SO₄ and concentrated. The collected crudeproduct was dissolved in dry DCM (85 ml) under nitrogen gas. Triphosgenewere added and the reaction mixture was cooled to 0° C., and Et₃N wasadded drop wise. The reaction mixture was stirred overnight at roomtemperature. Thin layer chromatography showed that the reaction wascompleted. The crude reaction produce was dissolved in dry DMF undernitrogen atmosphere, and 2-((2-(diethylamino)ethyl)thio)acetic acid(3.93 mmol, 751 mg) and EDC HCl (5.45 mmol, 1.0 g) were added. Thereaction mixture was stirred for overnight at room temperature. Thereaction was quenched with water (3 ml) and excess DMF was removed undervacuum at 25° C. The reaction product was diluted with water and aqueouslayer was extracted thrice with DCM (20 ml). The combined organic layerswere washed with brine solution and dried over anhydrous Na₂SO₄ .Concentration of the solvent gave a crude mass of 2 gram. Afterpurification by silica gel column (100-200 mesh) using 3% MeOH/DCM., theyield was 1.2 grams (76%).

Example 14 Synthesis of ATX-009

Di((Z)-non-2-en-1-yl) 8,8′((tertbutoxycarbonyl)azanediyl) dioctanoate(13.85 mmol, 9 grams) was dissolved in dry DCM (20 ml). TFA was added at0° C. to initiate a reaction. The reaction temperature was slowlyallowed to warm to room temperature over for 30 minutes with stirring.Thin layer chromatography showed that the reaction was completed. Thereaction product was concentrated under vacuum at 40° C. and the cruderesidue was diluted with DCM, and washed with a 10% NaHCO₃ solution. Theaqueous layer was re-extracted with DCM, and the combined organic layerswere washed with brine solution, dried over Na₂SO₄ and concentrated.Di((Z)-non-2-en-1-yl)8,8′-azanediyldioctanoate (0.909 mmol, 500 mg) wasdissolved in dry DCM (20 ml) under nitrogen atmosphere. Triphosgene wereadded and the reaction mixture was cooled to 0° C., and Et₃N was addeddrop wise. The reaction mixture was stirred overnight at roomtemperature. Thin layer chromatography showed that the reaction wascompleted. DCM solvent was removed from the reaction mass bydistillation under nitrogen atmosphere.2-(ethyl(methyl)amino)ethane-1-thiol hydrochloride (4.575 mmol, 715 mg)was dissolved in DMF (7 ml) and tetrahydrofuran (THF) (5 ml), and wasadded drop wise to the sodium hydride suspension in THF at 0° C. Thereaction mixture was then stirred overnight at room temperature. Thinlayer chromatography showed that the reaction was completed. Thereaction product was diluted with ethyl acetate and cold water. Thereaction was neutralized with 5% HCl (9 ml), and the organic layer wasseparated. The aqueous layer was re-extracted with ethyl acetate (EtOAc)(20 ml), washed in cold water and brine, and the combined organic layerswere washed dried over anhydrous Na₂SO₄. Concentration of the solventgave 1 gram or crude product. The compound was purified by silica gelcolumn (100-200 mesh) using 3% MeOH/DCM to yield 100 mg.

Example 15 Synthesis of ATX-010

Di((Z)-non-2-en-1-yl) 8,8′((tertbutoxycarbonyl)azanediyl) dioctanoate(3.079 mmol, 2 g) was dissolved in dry DCM (20 ml). TFA was added at 0°C. to initiate a reaction. The reaction temperature was slowly allowedto warm to room temperature over for 30 minutes with stirring. Thinlayer chromatography showed that the reaction was completed. Thereaction product was concentrated under vacuum at 40° C. and the cruderesidue was diluted with DCM, and washed with a 10% NaHCO₃ solution. Theaqueous layer was re-extracted with DCM, and the combined organic layerswere washed with brine solution, dried over Na₂SO₄ and concentrated. Thecollected crude product was dissolved in dry DCM (20 ml) under nitrogengas. Triphosgene (14.55 mmol, 4.32 g) was added and the reaction mixturewas cooled to 0° C., and Et₃N was added drop wise. The reaction mixturewas stirred overnight at room temperature. Thin layer chromatographyshowed that the reaction was completed. DCM solvent was removed from thereaction mass by distillation under N₂. The reaction product was cooledto 0° C., diluted with DCM (20 ml), and 2-(dimethylamino)ethanethiol HCl(0.063 mol, 8.3 g) was added, followed by Et₃N (dry). The reactionmixture was then stirred overnight at room temperature. Thin layerchromatography showed that the reaction was completed. The reactionproduct was diluted with 0.3 M HCl solution (20 ml), and the organiclayer was separated. The aqueous layer was re-extracted with DCM, andthe combined organic layers were washed with 10% K₂CO₃ aqueous solution20 ml) and dried over anhydrous Na₂SO₄. Concentration of the solventgave a crude mass of 10 gram. The crude compound was purified by silicagel column (100-200 mesh) using 3% MeOH/DCM. The yield was 1.4 g (75%)

Example 16 Synthesis of ATX-011 to ATX-030 and ATX-32

The synthesis of ATX-011 to ATX-30 follows the synthesis of Examples1-15, by substituting appropriate starting ingredients for syntheticreactions described therein.

Example 17 Synthesis of ATX-031

Example 19 In Vivo Mouse Factor VII Silencing

Using a liver-directed in vivo screen of the liposome libraries, aseries of compounds were tested that facilitate high levels of siRNAmediated gene silencing in hepatocytes, the cells comprising the liverparenchyma. Factor VII, a blood clotting factor, is a suitable targetgene for assaying functional siRNA delivery to liver. Because thisfactor is produced specifically in hepatocytes, gene silencing indicatessuccessful delivery to parenchyma, as opposed to delivery to the cellsof the reticulo-endothelial system (e.g., Kupffer cells). Furthermore,Factor VII is a secreted protein that can be readily measured in serum,obviating the need to euthanize animals. Silencing at the mRNA level canbe readily determined by measuring levels of protein. This is becausethe protein's short half-life (2-5 hour). C57BL/6 mice (Charles RiverLabs) received either saline or siRNA in liposome formulations via tailvein injection at a volume of 0.006 ml/g. At 48 h after administration,animals were anesthetized by isofluorane inhalation and blood wascollected into serum separator tubes by retroorbital bleed. Serum levelsof Factor VII protein were determined in samples using a chromogenicassay (Biophen FVII, Aniara Corporation) according to manufacturers'protocols. A standard curve was generated using serum collected fromsaline-treated animals.

Compositions with siRNA directed to Factor VIII were formulated withATX-001, ATX-002, ATX-003, and ATX-547, and comparator samples NCl andMC3 (Alnylam). These were injected into animals at 0.3 mg/kg and at 1mg/kg. The siRNA encapsulated by MC3 (0.3 mg/kg), NCl (0.3 mg/kg),ATX-547 (0.3 mg/kg), ATX-001 (0.3 and 1.0 mg/kg), ATX-002 (0.3 and 1.0mg/kg), and ATX-003 (0.3 and 1.0 mg/kg) was measured for the ability toknockdown Factor VII in mouse plasma following administration of thesiRNA formulation to C57BL6 mice. The results showed that ATX-001 andATX-002 were most effective at 0.3 mg/kg, compared to controls (FIGS. 1and 2).

The siRNA encapsulated MC3 (0.3 and 1.5 mg/kg), NCl (0.3 mg/kg), ATX-547(0.1 and 0.3 mg/kg), ATX-004 (0.3), ATX-006 (0.3 and 1.0 mg/kg), ATX-010(0.3 mg/kg), and ATX-001 (0.3 and 1.5 mg/kg), was measured for FactorVII knockdown in mouse plasma following administration of the siRNAformulation to C57BL6 mice. The results showed that ATX-001 and ATX-010were most effective (FIGS. 3 and 4). The knockdown activity of theexemplary compounds is shown for 0.3 mg/kg or at 0.05 mg/kg for ATX-018,ATX-019, and ATX-020 (Table 1).

What is claimed:
 1. A compound of formula I

wherein R₁ and R₂ are the same or different, each a linear or branchedalkyl of 1, 2, 3, 4, 5, 6, 7, 8, or 9 carbons, an alkenyl or alkynyl of2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 carbons, or cholesteryl, L₁ and L₂ arethe same or different, each a linear alkylene or alkenylene of 3 or 4carbons, X_(i) is —CO—O— whereby -L₂-CO—O—R₂ is formed, X₂ is S or O, X₃is —CO—O— whereby -L₁-CO—O—R₁ is formed, L₃ is a linear or branchedalkylene of 1, 2, 3, 4, 5 or 6 carbons, R₃ is a linear or branchedalkylene of 1, 2, 3, 4, 5 or 6 carbons, and R₄ and R₅ are the same ordifferent, each hydrogen or a linear or branched alkyl of 1, 2, 3, 4, 5or 6 carbons; or a pharmaceutically acceptable salt thereof.
 2. Thecompound of claim 1, wherein L₁ and L₂ are the same.
 3. The compound ofclaim 2, wherein L₁ and L₂ are propylene.
 4. The compound of claim 1,wherein R₁ and R₂ are the same.
 5. The compound of claim 1, wherein L₃is a methylene.
 6. The compound of claim 1 consisting of the compound offormula ATX-0030


7. A compound of formula 2

wherein R₁ and R₂ are the same or different, each a linear or branchedalkyl of 1, 2, 3, 4, 5, 6, 7, 8, or 9 carbons, an alkenyl or alkynyl of2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 carbons, or cholesteryl, L₁ and L₂ arethe same or different, each a linear alkylene or alkenylene of 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbons, X₁ is —CO—O—whereby -L₂-CO—O—R₂ is formed, X₂ is S or O, X₃ is —CO—O— whereby-L₁-CO—O—R₁ is formed, L₃ is a linear or branched alkylene of 1, 2, 3,4, 5 or 6 carbons, R₃ is a linear or branched alkylene of 1, 2, 3, 4, 5or 6 carbons, and R₄ and R₅ are the same or different, each a hydrogen,or a linear or branched alkyl of 1, 2, 3, 4, 5 or 6 carbons; or apharmaceutically acceptable salt thereof.
 8. The compound of claim 7,wherein L₃ is methylene.
 9. The compound of claim 7, wherein R₄ and R₅are independently methyl or ethyl.
 10. The compound of claim 7, whereinR₃ is ethylene or propylene.
 11. The compound of claim 7, wherein X₂ isS.
 12. The compound of claim 7, wherein R₁ and R₂ are the same.
 13. Thecompound of claim 12, wherein R₁ and R₂ are an alkyl of 1, 2, or 3carbons.
 14. The compound of claim 12, wherein R₁ and R₂ are an alkenylof 9, 10, or 11 carbons.
 15. The compound of claim 7, wherein L₁ and L₂are the same.
 16. The compound of claim 15, wherein L₁ and L₂ are analkylene of 3, 4, 5, 6, or 7 carbons.
 17. The compound of claim 15,wherein L₁ and L₂ are an alkenylene of 13, 14, or 15 carbons.
 18. Thecompound of claim 7, wherein X₂ is O.
 19. The compound of claim 7,wherein L₃ is a methylene.
 20. The compound of claim 7, selected fromATX-001, ATX-004, ATX-005, ATX-006, ATX-007, ATX-008, ATX-014, ATX-016,ATX-021, ATX-021, and ATX-027.